The present invention relates to optical instruments and, more particularly, to instruments for distinguishing particles according to optical effects that occur when the particles pass illuminated locations. A major objective of the present invention is to provide for reduced fluorescence crosstalk in a multi-laser fluorescence analyzer.
Testing laboratories need to be able to detect the presence of certain entities, e.g., antigens, that can be difficult to detect directly. In some cases, the entities can be tagged with fluorochromes which are detectable. For example, the antibody for an antigen can be derivatized with a fluorochrome. The derivatized antibody can be mixed with a blood sample. To the extent an antigen is present in a cell, the derivatized antibody binds to it rendering it fluorescent. The tagged cells can be introduced into a cytometry system, wherein they can then be illuminated with monochromatic radiation, e.g., from a laser, that excites the fluorochrome. A photodetector can then detect the intensity of the fluorescent emissions.
It is often necessary to identify cells with a particular combination of antigens. To this end, several antibodies can be derivatized with respective fluorochromes; the derivatized antibodies are mixed with the blood sample under conditions sufficient for the antibodies to bind with the respective antigens. The cells are then illuminated and the resulting fluorescent emissions detected and measured.
To the extent possible, the fluorochromes are selected to have distinct emission spectra. A typical fluorescent analyzer can include a blue laser to excite fluorochromes that emit green, yellow, and red light, respectively. Dichroic mirrors or other wavelength-dispersive elements can split the emissions into green, yellow and red beams that are directed to respective photodetectors. In practice, it is difficult to distinguish more than three fluorochromes by wavelength alone due to overlap in emission spectra.
The number of distinguishable fluorochromes can be increased by using more than one excitation wavelength. This approach takes advantage of the fact that fluorochromes differ not only in their emission spectra, but in their excitation spectra. In an ideal case, two fluorochromes with nonoverlapping excitation spectra could be distinguished even where the emission spectra were identical. The distinction could be achieved by illuminating the fluorochromes at different times with two lasers, each selected to excite only a respective one of the fluorochromes. The resulting emissions would appear as two distinct signal pulses in the output of a single photodetector.
This approach is implemented in the context of a flow cytometry system by illuminating different locations along the flow tube with different laser wavelengths, each of which preferentially excites a respective fluorochrome. Tagged cells are made to flow past the two locations sufficiently infrequently that, usually, only one location is occupied at any given time. When a cell is at the first location, a photodetector pulse corresponds to the first fluorochrome; when later the cell is at a second location, a photodetector pulse corresponds to the second fluorochrome. Since the fluorochromes are distinguishable in the time domain, a single photodetector can be used to detect the emissions for both fluorochromes.
Cytometry systems in which a single photodetector is used to detect emissions resulting from spatially and wavelength separated excitations are disclosed or suggested by: 1) Donna J. Arndt-Jovin, Brian G. Grimwade, and Thomas M. Jovin, "A Dual Laser Flow Sorter Utilizing a CW Pumped Dye Laser" Cytometry Vol. 1, No. 2, 1980, pp. 127-131; 2) Eugene Hamori, Donna J. Arndt-Jovin, Brian G. Grimwade and Thomas M. Jovin, "Selection of Viable Cells with Known DNA Content" Cytometry, Vol. 1, No. 2, 1980, p. 132-1352; and 3) Julianne Meyne, Marty F. Bartholdi, Gayle Travis, and L. Scott Cram, "Counterstaining Human Chromosomes for Flow Karyology", Cytometry, No. 5, 1984, pp. 580-583.
In practice, each fluorochrome may be weakly excited by the excitation frequency that strongly excites the other fluorochrome. Thus, each fluorochrome can contribute "crosstalk" to the electrical pulse occurring in the time slot allotted to the other fluorochrome, resulting in erroneous readings. This crosstalk can be reduced by using two photodetectors, each positioned to receive only emissions from one of the excitation locations. However, this incurs the considerable expense and bulk associated with an additional photodetector. What is needed is a system that reduces crosstalk between fluorochromes that have substantially overlapping emission spectra and slightly overlapping excitation spectra without requiring an additional photodetector.